* Bruce Ivins’ co-authors can explain the rabbit and other animal protocols that applied to the subcutaneous challenges in B3 in Building 1425 conducted in September and October 2001.
Posted by DXer on November 9, 2011
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CASE CLOSED ... what really happened in the 2001 anthrax attacks? 
DXer said
Thousands of Monkeys Made to Suffer Post-9/11: Report
BY LAUREN WALKER 7/7/15 AT 3:59 PM
http://www.newsweek.com/thousands-monkeys-made-suffer-post-911-351067
DXer said
By FOIA requested dated November 7, 2001, I wrote:
“To clarify my earlier request for the rabbit protocol that was being forwarded by Dr. Ivins in an earlier referenced email, note that based on this additional email, dated May 1, 2002, I believe the computer document that I request is indicated to be titled “ADDENDUM rabbit protocol03.doc”.
I am thinking therefore that the other two relevant rabbit protocols dating to Fall 2001 of keen interest are titled rabbitprotocol02.doc and rabbitprotocol.doc.
I don’t know if this helps but it may help an IT person locate the document(s). ….”
We want the rabbit protocol ADDENDUM rabbit protocol03.doc.
DXer said
As Norm Covert can explain, there was a committee that reviewed protocols and imposed additional requirements deemed necessary to avoid the unnecessary suffering of animals. Requirements were evolving.
DXer said
Dr. Fellows would know whether there were 3Xs or 2X checks a day — as would Mr. Bassett for one.
We otherwise need to await an executed animal protocol that confirms the third check at night.
DXer said
Although I am not scientifically trained, there are many experts who could address what is involved with bacteremia studies involving rabbits at USAMRIID and elsewhere.
Aerosolized Bacillus anthracis Infection in New Zealand White Rabbits: Natural History and Intravenous Levofloxacin Treatment
Steven B Yee,1* Joshua M Hatkin,4 David N Dyer,1 Steven A Orr,2,3 M Louise M Pitt1
1Center for Aerobiological Sciences United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland;
2Office of Regulated Studies, United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland;
3Clinical Research Management, Hinckley, Ohio;
4Pennsylvania Veterinary Diagnostic Laboratory, Harrisburg, Pennsylvania.
*Corresponding author. Email: steven.yee@us.army.mil
Animals.
Bordetella-free SPF male and female New Zealand white rabbits (2.2 to 3.5 kg) were acquired from Charles River Laboratories (Pointe-Claire, Quebec, Canada). Animals were housed in individual cages and were acclimated to a 12:12-h light:dark cycle in a temperature- and humidity-controlled, SPF environment. Rabbits were maintained according to facility standard operating procedures, with food and water provided ad libitum. All rabbits were acclimated to the facility for at least 7 d before surgical implantation of a venous access port (Solomon Scientific, San Antonio, TX) to facilitate frequent phlebotomy sampling. In addition, rabbits used in the natural history study were implanted with a radiotelemetry device (model TA10TA-D70; Data Sciences International, St Paul, MN) to measure body temperature. Before inclusion in any study, rabbits were allowed to recover from surgery for 1 wk and were determined to be clinically healthy.
Research was conducted in compliance with the Animal Welfare Act2 and other applicable federal statutes and regulations related to animals and experiments involving animals and conformed to the guidelines for humane treatment set by the Guide for the Care and Use of Laboratory Animals.20The United States Army Medical Research Institute of Infectious Disease facility is fully AAALAC-accredited. All research was conducted under approved protocols by the Institutional Animal Care and Use Committee at the United States Army Medical Research Institute of Infectious Disease.
***
Spore preparation.
As described previously,39 in a Biologic Safety Level 3 laboratory, B. anthracis (Ames strain) spores were produced in flask cultures containing Leighton and Doi medium.27 After centrifugation, the spore pellet was washed in sterile water and purified on gradients of 60% Hypaque-76 (GE Healthcare, Piscataway, NJ). Spores were stored in 1% phenol at 4 °C until use. Phenol was removed before aerosolization and spores were resuspended in sterile water and heat-shocked at 60 °C for 45 min.
***
Aerosolized B. anthracis challenges occurred in a class III biological safety cabinet situated within an approved, restricted access Biological Safety Level 3 laboratory at the United States Army Medical Research Institute of Infectious Disease.
***
Natural history study.
Starting at 9 h after B. anthracis challenge and continuing at 3- or 6-h intervals thereafter, blood was collected by means of the vascular access port until either 96 h after exposure or the rabbit had succumbed to the disease or became moribund and was euthanized. Each blood sample was subdivided for CBC, bacteremia, and antigenemia analysis. Body temperature was monitored continuously and clinical observations made until B. anthracis-challenged rabbits succumbed or were euthanized.
***
Bacteremia.
Whole-blood samples were collected in Wampole isolator microbial tubes (Inverness Medical, Princeton, NJ) and cultured on tryptic soy agar. Plates (in duplicate) were incubated for 18 to 24 h at 37 °C and evaluated for colonies at 24 h after sampling.
Antigenemia.
PA was determined qualitatively from whole blood by ECL immunoassay (Bioveris M-Series M1M System Analyzer; Bioveris, Gaithersburg, MD), as previously described.39 Three PA-specific monoclonal antibodies were used as a capture-antibody mix.11,29,39 All samples were analyzed in duplicate. Positive controls consisted of naïve rabbit whole blood spiked with PA (Critical Reagents Program, Aberdeen Proving Grounds Edgewood Area, Chesterton, MD), whereas negative matrix controls consisted of whole blood only. Samples were considered ECL positive, and thereby antigenemic, if the signal:noise ratio was ≥ 1.2 times the average of the negative matrix controls. The ECL limit of detection for PA-spiked whole blood was less than 2 ng/mL. Antigenemia results by ECL assay were determined within 1 h of blood collection.
Levofloxacin efficacy.
Starting 15 h after challenge with B. anthracis and continuing at 3-h intervals thereafter, blood was collected from the vascular access port until rabbits were determined to be antigenemic by ECL immunoassay or 30 h after exposure. Blood was collected every 24 h thereafter. Blood samples were subdivided for antigenemia and bacteremia analysis. After antigenemia determination, B. anthracis-challenged rabbits intravenously received either saline vehicle (control) or levofloxacin (total daily dose, 12.5 or 25 mg/kg; Ortho Pharmaceutical, Raritan, NJ). Vehicle or levofloxacin was administered twice daily, in half-total daily doses at 12-h intervals. Efficacy end-point was survival time, defined as the time from B. anthracis challenge to 28 d after challenge. Clinical observations were made at least twice daily until the rabbit succumbed to the disease or was euthanized.
***
Development of antigenemia and bacteremia in B. anthracis-challenged rabbits.
Both antigenemia and bacteremia developed before the occurrence of pyrexia and inversion of the heterophil:lymphocyte ratio (Table 1 and Figure 1). Heterophil:lymphocyte inversion is an indicator of infection.46 The presence of PA in rabbit blood (that is, antigenemia) was detected starting 24.5 ± 1.4 h (range, 15 to 30 h) after B. anthracis challenge. PA detection by ECL immunoassay was simple and reproducible,39 with results obtained within 1 h of blood collection. Similar to antigenemia, bacteremia developed between 15 and 30 h after B. anthracis challenge, for a mean occurrence time of 23.1 ± 0.2 h. However, bacteremia determination by culture was considerably more time-intensive than was antigenemia detection, with bacteremia results obtained within 18 to 24 h of blood collection. Qualitative ECL signal:noise values positively correlated with bacterial load (r = 0.923, P < 0.0000002). The ECL signal:noise level and bacterial load remained elevated until B. anthracis-challenged animals succumbed to inhalational anthrax (data not shown). Interestingly, neither the development of antigenemia nor bacteremia correlated with either the presented dose or postchallenge survival time. Although both antigenemia and bacteremia positively correlated with the occurrence of pyrexia (r = 0.757, P = 0.0112 and r = 0.806, P = 0.00866, respectively), only bacteremia correlated with inversion of the heterophil:lymphocyte ratio (r = 0.712, P = 0.0315). No sex-associated differences were observed in the development of antigenemia or bacteremia after challenge.
***
Therapeutic window for B. anthracis-challenged rabbits.
Given antigenemia as the biomarker of systemic infection for treatment initiation, postantigenemia survival time in B. anthracis-challenged rabbits was approximately 22.7 ± 3.5 h, ranging from 8 to 40 h after antigenemia (Table 1 and Figure 2). This result suggests a relatively narrow window of opportunity for treatment.
***
Antigenemia and bacteremia development preceded the occurrence of pyrexia and heterophil:lymphocyte inversion (Table 1) in rabbits challenged with aerosolized B. anthracis. Bacteremia is considered the ‘gold standard’ for diagnosis of systemic infection. However, recent studies involving inhalational anthrax have determined that the detection of PA is a comparable, reliable biomarker of systemic infection in rabbits and nonhuman primates.24,32,34,39 Bacterial load in the present study positively correlated with antigenemia, supporting the premise that antigenemia may be used as a surrogate for bacteremia to estimate severity of disease.24 Despite the development of bacteremia and antigenemia at relatively the same time after B. anthracischallenge, antigenemia determination by ECL immunoassay is considerably more rapid (that is, within approximately 1 h) than the traditional 18- to 24-h culture needed to determine bacteremia. ECL immunoassay therefore allows for timely, appropriate therapeutic intervention. Hence, antigenemia serves an early biomarker of systemic infection.